泳池與農田棘阿米巴原蟲定性研究

Characterization of Acanthamoeba Isolated from Swimming Pools and Farmlands

10.6342/NTU.2012.01657

張維珍

棘阿米巴原蟲 ； 角膜炎 ； 肉芽腫性阿米巴腦膜炎 ； 致病性 ； PCR ； 環境因子 ； 生物指標 ； 基因型 ； 耐熱性 ； 耐滲透壓 ； Acanthamoeba ； keratitis ； granulomatous amebic encephalitis ； pathogenicity ； PCR ； environmental factors ； biological indicator ； genotype ； thermotolerance ； osmotolerance

臺灣大學環境衛生研究所學位論文

2012年

碩士

張靜文

英文

棘阿米巴原蟲（Acanthamoeba）為自營性阿米巴原蟲，廣泛存在於自然及人工環境的水體、土壤與空氣中。棘阿米巴原蟲為伺機性的致病微生物，可導致嚴重威脅視力的棘阿米巴角膜炎、以及九成高致死率的肉芽腫性阿米巴腦膜炎。棘阿米巴原蟲感染的機制取決於棘阿米巴原蟲致病性、宿主與環境因子。此外，棘阿米巴原蟲不只感染人類，也是其他致病菌的自然界宿主。 本研究利用形態學和分子生物學方法檢測來自台北市6個游泳池之12件游泳池樣本、以及來自6個屏東縣恆春鎮和5個屏東縣車城鎮洋蔥田農田之13件土壤與14件空氣樣本，鑑定其中是否有棘阿米巴原蟲，並利用基因型分析、耐熱性及耐滲透壓測試，評估分離的棘阿米巴原蟲是否具潛在致病性。另收集水體pH、水溫、導電度、濁度、硬度、溶氧、餘氯、鹽度、總溶解固體及異營性總細菌濃度，測量土壤pH和、含含量及異營性總細菌濃度，以及採樣時空氣溫度、風速和相對濕度，將環境因子特性與棘阿米巴原蟲基因型、耐熱及耐滲透壓結果交叉比較，分析環境因子是否影響棘阿米巴原蟲之潛在致病性。 結果發現，6個游泳池水樣檢出率為100%，13件屏東縣洋蔥田土壤檢出率亦為100%，其中14件空氣樣本中只有3件檢出棘阿米巴原蟲，其檢出率為21.43%。本研究共分離42株棘阿米巴原蟲，其中泳池水樣分離出26株、土壤為13株、空氣則是3株。在泳池水樣的26株皆為Acanthamoeba polyphaga，且皆屬於基因型T4。其中12株進行耐熱性和滲透壓測試，耐熱結果顯示除了內湖運動中心分離的一株不能耐熱37oC外，其餘11株都可耐熱37oC，但此12株都無法耐熱於42oC及52oC下；至於滲透壓結果顯示，12株泳池分離菌株在1M mannitol的培養基上都能生長。 至於在土壤分離之13株棘阿米巴原蟲，6株為Acanthamoeba polyphaga，屬於基因型T4，另外7株為Acanthamoeba lenticulata，屬於基因型T5。將此13株進行耐熱性和滲透壓測試，結果顯示7株可耐熱於52oC，2株耐熱於42oC，4株則僅能耐熱至37oC；滲透壓結果顯示所有13株皆無法生長在1 M mannitol的培養基，但都能在0.5 M mannitol的培養基中生長。 對於分離自空氣樣本的3株棘阿米巴原蟲，2株為Acanthamoeba polyphaga，屬於基因型T4，另外1株為Acanthamoeba species，屬於基因型T15。將此3株進行耐熱性和滲透壓測試，結果顯示，除恆春農田分離的一株棘阿米巴原蟲不能耐熱37oC外，另2株均可耐熱37oC，但此3株都不能耐熱至42oC與52oC；滲透壓結果顯示，有一株棘阿米巴原蟲能在0.5 M mannitol 與1M mannitol下生長，一株棘阿米巴原蟲僅能生長於0.5 M mannitol的培養基，一株則無法在0.5 M mannitol和1 M mannitol的培養基上生長。 依據基因型、耐熱性和滲透壓結果顯示，泳池分離出來的棘阿米巴原蟲均屬T4，且一株不能在37oC生長，其餘均能在37oC與滲透壓1 M mannitol下生長；而農田土壤及空氣分離之16株棘阿米巴原蟲大多數無法在1M mannitol的環境下生長，這顯示泳池中之棘阿米巴原蟲一旦有機會接觸到人體的眼睛，多可忍受人體體溫與淚液之高滲漏壓。意謂泳池內棘阿米巴原蟲可能具有眼睛角膜之致病風險，然土壤分離之部分菌株可耐高溫，仍宜關切其潛在風險。

Acanthamoeba is free-living amoebae and ubiquitous in a wide variety of natural habitats and human-made environments, including water, soil, and air, and a opportunistically pathogenic organism that can cause a severe sight-threatening Acanthamoeba keratitis (AK), and a fatal infection of the central nervous system (granulomatous amoebic encephalitis, GAE), mortality rate can up to 90%. The mechanisms associated with the pathogenesis of Acanthamoeba tend to be highly complex, depending on parasite, host and the environmental factors. Besides, these amoebae are not only infective to human but also the hosts for other pathogenic bacteria to multiply in the environments. In this study, we isolated and characterized Acanthamoeba from water, soils, and air in the effort to determine the presence and potentially pathogenic Acanthamoeba. There were 12 water samples from 6 chlorinated swimming pools in Taipei city, 13 soil samples and 14 air samples were from 6 onion farmlands in Hengchun Township, Pingtung County and 5 onion farmlands in Checheng Township, Pingtung County. The samples were then followed by morphology and molecular identification, phylogenetic and physiologically assays including thermotolerance and osmotolerance characterization. Environmental factors in water samples including pH, water temperature, conductivity, turbidity, hardness, dissolved oxygen, free chlorine, salinity, total dissolved solids and heterotrophic plate count. Water content, temperature and heterotrophic plate count in soil samples. Temperature, wind velocity and relative humidity in air were measured in order to characterize the sampling sites and to determine how these factors affecting the potentially pathogenicity of isolated Acanthamoeba by genotyping, thermotolerance and osmotolerance. In the present study, we discovered Acanthamoeba in six swimming pools (100%), 13 onion farmlands (100%), and only three out of 14 air samples (21.43%). Total 42 isolates of Acanthamoeba are isolated, 26 isolates from swimming pools, 13 isolates from soil, and three isolates from air samples. The 26 isolates in swimming pools are belong to Acanthamoeba polyphaga, and genotype T4, there are 12 isolates taken into thermotolerance and osmotolerance among all, only one isolate from NEIHU swimming pool cannot growth under 37oC, and 12 isolates cannot growth under 42oC and 52oC, in osmotolerance, 12 isolates can tolerant and growth up to 1 M mannitol. In 13 soil isolates, six isolates belong to Acanthamoeba polyphaga and genotype T4, and the rest 7 isolates belong to Acanthamoeba lenticulata, genotype T5, all 13 isolates were testing for the thermotolerance and osmotolerance, 7 isolates can tolerant up to 52oC, two isolates growth under 42oC, and only four isolates growth at 37oC, and 13 isolates cannot growth at 1 M mannitol, but can growth at 0.5 M mannitol. In three air isolates, two isolates belong to Acanthamoeba polyphaga and genotype T4, one isolate belongs to Acanthamoeba species, genotype T15, all three isolates were testing for the thermotolerance and osmotolerance, the 2 isolates can growth at 37oC besides isolate in HENGCHUN onion farmlands 4, which cannot growth at 37oC, three isolates cannot growth under 42oC and 52oC, when it comes to osmotolerance, only one isolate can growth under 0.5 M mannitol and 1 M mannitol, one isolate can growth under 0.5 M mannitol, and the other isolate cannot growth neither 1 M mannitol nor 0.5 M mannitol. The pathogenicity based on genotyping, thermotolerance and osmotolerance revealed the all isolates from swimming pools in present study are genotype T4 and among the 12 isolates with thermotolerance and osmotolerance, 11 isolates can grew at 37oC and 1 M mannitol. Once the pool isolates contact with human eyes, they can tolerate body temperatures and osmolarity of tear film. Most of 16 isolates from soil and air samples indicated weak pathogenicity since these isolates cannot growth under 1 M mannitol. Acanthamoeba isolates from swimming pools indicated higher potentially pathogenicity while the potential risk on isolates from soils since isolates can withstand at higher temperature.

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